This invention relates to cuvettes in which reactions are undertaken to amplify and detect nucleic acids, using PCR technology, without exposing the environment to amplified nucleic acid.
Polymerase chain reaction (PCR) technology is only one of several techniques that permit nucleic acid material, such as DNA, often extracted from as little as a single cell, to be amplified to hundreds of millions of copies. This is important since prior to PCR technology it was virtually impossible to detect a single DNA strand. However, when a single DNA strand, such as the DNA of the human immunodeficiency virus (HIV, otherwise known to cause AIDS), is added to amplifying reagents that will amplify the DNA of choice, hundreds of millions of copies of that DNA can be obtained in a relatively short time. Technology further allows for the detection of the amplified nucleic acid material (DNA for example), using probes that hybridize to the amplified material of choice, such probes in turn either being immobilized or immobilizable to a solid support, such as a filter membrane, and/or being labeled for detection using enzymes or other moieties.
Conventionally, this has been done by amplifying the nucleic acid material in a stoppered plastic container until the desired number of copies have been formed. Thereafter, the container is reopened, such as by unstoppering, and either the amplified copies are withdrawn and transferred to detection apparatus, or detecting reagents can be added to the container used for the amplification, so that detection is done in the same container.
It has been discovered that such a technique is unsatisfactory for convenient and widespread use of, e.g., PCR technology, because aerosols are produced in the act of unstoppering and/or transfer of fluids. Such aerosols contain a few of the amplified nucleic acid material, e.g., DNA. The aerosols then proceed to disperse within the environment. Normally, such few molecules in the environment are not of great concern. However, only one DNA molecule is needed to ruin by contamination other amplifying containers yet to be used for detection. That is, if the errant DNA molecule floats into or is carried, inadvertently, by an operator to another amplifying container yet to be used, that one molecule is all that is needed to provide the DNA needed for the next amplification. Needless to say, if the point of the next test is to see if a particular DNA is present (e.g., for HIV), and it is detected only because of the errant DNA and not that of the patient, the test is ruined. Thus, the very power of DNA amplification becomes the source of potential ruin of the tests. As a matter of fact, an entire lab has been proven to be contaminated by the unstoppering of just a few containers in which the sample has already been amplified. Although such a problem might be avoidable by using highly skilled and trained personnel who painstakingly minimize the aerosols produced, the need for such labor makes the technology impractical for general use.
Thus, it has been a problem prior to this invention to provide apparatus and a method for amplifying and detecting nucleic acid material, without contaminating the surrounding environment.
Yet another problem has been, prior to this invention, to automate the detection steps, that is, minimize the need for operator intervention. The need to transfer amplified nucleic acid material or to add detection reagents makes such automation difficult.
The above problems are addressed by an apparatus and a method that solve the above-mentioned needs. The invention is based upon the realization that the contamination can be prevented by confining the amplifying reagents and amplified nucleic acid in the cuvette so that it is impossible for any amplified nucleic acid molecules to escape.
More specifically, in accord with one aspect of the invention, there is provided a cuvette for the amplification and detection of DNA, the cuvette including a plurality of compartments including a) means for allowing DNA amplification, the allowing means including a reaction compartment and means adjacent to the reaction compartment for permitting active or passive cycling of the contents of the reaction compartment through a temperature range of from about 30xc2x0 C. to about 95xc2x0 C.; b) means for providing liquid interconnection between the compartments by pressurizing the liquid; and c) means for trapping and holding DNA at a detection site for detection, including a detection material capable of generating a detectable signal. The cuvette is improved in that some of the compartments contain the detection material and the reagent in unreacted form in storage, while the cuvette is free of DNA sample, whereby the cuvette need not be reopened between DNA amplification and detection.
In accord with another aspect of the invention, there is provided a closed, disposable cuvette for carrying out amplification and detection of nucleic acid material, comprising: a plurality of compartments including a reaction compartment, means permitting active or passive cycling of the contents of the reaction compartment through a temperature range; at least one detection material being present in at least one of the compartments; and means for fluidly interconnecting the compartments in prescribed order when pressure is applied to the contents of a compartment. The cuvette is improved in that the compartments all are closed to fluid flow to locations outside of the container and said reaction compartment contains nucleic acid material and unreacted amplifying reagents; at least one of the compartments including means at a detection site therein for immobilizing the nucleic acid material for detection after amplification, so that detection of amplified nucleic acid material occurs without contamination of other containers or apparatus by the amplified nucleic acid material. The result is that detection of amplified nucleic acid material occurs without contamination of other containers or apparatus by the amplified nucleic acid material.
In accordance with still another aspect of the invention, there is provided a closed cuvette as described in the previous paragraph, wherein the reagent contents of the reaction compartment comprise polymerase enzyme, primer nucleic acids and deoxyribonucleotides.
In accord with yet another aspect of the invention, there is provided an apparatus for amplifying and detecting DNA, comprising a cuvette containing i) a plurality of compartments and means for interconnecting each of them to at least one other compartment, the compartments including a) at least one reaction compartment for amplifying DNA strands, b) at least one detection compartment for detecting amplified DNA and including a detection site, and c) means for delivering a detection material to amplified DNA strands; ii) means for permitting active or passive cycling of the contents of the reaction compartment through a temperature range; and iii) liquid access means connected only to the at least one reaction compartment for allowing the injection into the reaction compartment of a sample DNA for amplifying; characterized in that the cuvette further includes iv) means sealing the cuvette against passage of DNA after sample DNA is injected; and the apparatus further includes means for moving at least the detection material and a DNA strand into the detection compartment and onto the detection site; so that once a DNA sample is injected into the compartments and the access aperture is closed, the fluid contents of the compartments are contained against contact by the operator and environment during the entire amplification and detection reaction.
In still another aspect of the invention, there is provided a method for amplifying and detecting nucleic acid material in a closed cuvette without allowing aerosols to exit therefrom to contaminate the environment, the method comprising the steps of a) injecting a sample of nucleic acid material into a cuvette comprising a plurality of compartments including a reaction compartment wherein amplifying reagents are present, and a storage compartment for use with a detection material, at least one of the compartments including a detection site, and means for interconnecting the compartments to provide fluid transfer; b) closing off permanently the portions of the cuvette containing the nucleic acid material to lock all nucleic acid into the cuvette; c) amplifying the nucleic acid material by cycling the cuvette through temperature changes preselected to cause the reagents to be effective; d) fluidly transferring amplified nucleic acid material from the reaction chamber to the detection site; e) fluidly transferring detection material to the detection site while keeping the cuvette closed; and f) detecting the amplified nucleic acid material at the detection site with the detection material, all while the nucleic acid material remains confined within the cuvette.
It is an advantageous feature of the invention that a cuvette is provided for amplifying nucleic acids that avoids the risk of contaminating the environment with amplified nucleic acid since it avoids reopening the area of the cuvette containing such nucleic acid.
It is a related advantageous feature of the invention that a cuvette is provided that can be used for such amplification by relatively unskilled labor.
It is another advantageous feature of the invention that such a cuvette is provided that is amenable to automated processing.
Other advantageous features will become apparent upon reference to the detailed description that follows, and the attached drawings.